The thalassemias represent a heterogeneous group of diseases, characterized by the absence or diminished synthesis of one or the other of the globin chains of hemoglobin A. In .alpha.-thalassemia, .alpha.-chain synthesis is decreased or absent; whereas in .beta.-thalassemia, .beta.-chain synthesis is diminished or absent. Numerous molecular defects account for the various thalassemias. The degree of clinical expression is generally dictated by the nature and severity of the underlying globin gene (DNA) defect. Thalassemia major (homozygous .beta.-thalassemia) defines the most severe variety of the disease. Thalassemia intermedia and thalassemia minor refer to the heterozygous state, generally associated with milder clinical manifestations.
Beta-thalassemia is an autosomal recessive disorder characterized by absent (.beta..sup.o) or decreased (.beta..sup.+) synthesis of the .beta.-globin chain. Thalassemia is found in almost all population and ethnic groups around the world. It has been estimated that 3% of the world's population or 150 million people carry .beta.-thalassemia genes. Indeed, it is among the most common genetic diseases in the world. Alpha thalassemia, the corresponding disorder of .alpha. hemoglobin chain is also of great prevalence, especially in the Orient.
By the next century, probably over 50% of the world population in certain groups will be at risk for the thalassemias. Thus, in world terms, the problem would be anticipated to increase with time. Because these predictions have tremendous medical, social and economic implications, particularly in developing nations, a major challenge to health professionals is the development of methodologies for molecular diagnosis of the thalassemia syndromes that are simple and reliable, especially those that do not depend on radioactivity. Such a strategy would allow for detection, as well as facilitate genetic counseling of carriers.
Traditional detection of .beta.-thalassemia has relied heavily on the hematological tests. With the more generalized use of electronic cell counters, the diagnosis is first suspected by the discovery of a low MCV and MCH on routine "complete" blood counts. Increased levels of HbA.sub.2 (to 4-6 percent) and/or increased HbF (up to 5-20 percent) demonstrated by quantitative hemoglobin electrophoresis supports the diagnosis. Unfortunately, the differential diagnosis between iron deficiency anemia and .beta.-thalassemia trait can be difficult in practice, if there are not reciprocal increases in HbA.sub.2 and/or HbF. Moreover, in the presence of concomitant iron-deficiency, HbA.sub.2 levels in .beta.-thalassemic individuals may fall into the normal range. Occasionally, the diagnosis of iron deficiency cannot be made on the basis of measurements of serum iron, iron binding capacity and/or the absence of stainable iron in the bone marrow. In these instances, the demonstration of a reduced .beta.-globin synthetic rate (compared to .alpha.-globin), generally employing .sup.3 H-leucine to analyze globin chain production in reticulocytes is required for a conclusive diagnosis. This procedure is cumbersome and not well-suited for large scale investigations.
Recently, with improvements in techniques in molecular biology and with the cloning of the human globin genes, cloning and sequencing the .beta. globin genes from a patient with suspect .beta. thalassemia (or the .alpha. globin gene from patients suspected to have alpha thalassemia) have become possible. More recently, it has been found that a number of point mutations that result in the thalassemic phenotype are genetically linked to specific restriction fragment length polymorphisms or haplotypes. Thus, by isolating DNA from peripheral tissues (usually leukocytes), one can perform restriction enzyme digestion of genomic DNA and Southern blotting to examine for the presence of specific haplotypes from which one can infer the particular thalassemic mutation. However, the linkage of different .beta.-globin gene cluster haplotypes with specific mutations giving rise to the .beta.-thalassemia is not definitive. Moreover, the isolation, cloning and sequencing of the defective .beta.-globin gene(s) in suspected carriers is time-consuming and not well suited for large population studies.